Icosahedral capsid assembly is a highly coordinated process involving sequential addition of multiple proteins, ultimately leading to an infectious virion of proper size and morphology. The long-term goal for this project is to achieve a mechanistic understanding of the protein:protein interactions involved in capsid assembly. The development of new anti-viral drugs is impeded by a lack of understanding of how viral capsid proteins are programmed to adopt the correct conformations to produce the correct assembly product. Capsid assembly will be investigated using bacteriophage P22, a model dsDNA virus. In phage P22, herpesvirus and many other dsDNA viruses, the capsid is formed from a coat protein having the ubiquitous HK97 fold. The initial assembly product is a procapsid (PC). Scaffolding protein (SP) directs proper assembly of coat protein (CP) to form PCs. SP also directs the incorporation of the portal protein complex, which is essential for genome encapsidation. Phage P22 provides an excellent model assembly system because complex in vivo processes are easily mimicked in vitro. The simple genetics and well-established biochemistry of phage P22 offers significant advantages as an assembly model over complex mammalian dsDNA viruses. Our central hypothesis is that specific weak protein:protein interactions regulate the assembly nucleation and elongation reactions in order to form proper procapsids and virions. In this granting period we will test our central hypothesis with the following aims. Aim 1. Define the mechanism of portal protein complex incorporation into PC. We hypothesize that SP controls portal protein incorporation during PC assembly through interaction with a conserved belt of hydrophobic residues on the surface of the portal rings. The portal protein is essential to form an infectious virion for the tailed phages, herpesviruses and adenoviruses. Though characterization of mutants in SP and portal protein, and the use of ssRNA aptamers specific for portal or SP, we will elucidate the mechanism of portal incorporation during assembly. Aim 2. Understand control of capsid morphology. We hypothesize specific CP conformational changes induced by SP control procapsid and capsid morphology. We will characterize the interaction by single molecule fluorescence methods. We will investigate how CP controls capsid morphology by characterizing CP mutants that change the size and shape of PCs. Aim 3. Understand how scaffolding protein functions in PC assembly. We hypothesize that SPs have intrinsically disordered segments to allow them to interact with the many protein partners required to assemble PCs. There is very little high-resolution information about their structures, either in solution or within PCs. We will use state-of-the-art NMR techniques combined with mutational analysis to characterize the structure of scaffolding proteins from phages P22 and Sf6.